Affibodies are a new class of engineered affinity proteins derived from staphyloccocal protein A (SPA) B domain. B domain is composed of 58 amino acids with a molecular mass of 6.5 ku and has a three-helical bundle structure. Thirteen amino acid positions in helix 1 and helix 2 of B domain can be randomly mutated to construct an affibody library without obvious compromise in its structural stability. Theoretically, this library contains affibodies specific to any given target. Using affinity panning, a specific affibody can be obtained. Affibody has some functional similarities with antibody. However, compared with antibody, affibody has some outstanding properties. Affibody can be obtained by in vitro selection and can be produced in large scale by chemical synthesis or prokaryotic expression. The smaller molecular size confers its higher tissue penetrativity and faster circulation clearance. Affibody has a higher physical and chemical stability than antibody. In addition, affibody can be cross-liked with or fusion co-expressed with reporter molecules such as fluorescent protein, biotin and so on. This class of engineered affinity proteins has been used in detection, separation, and purification of target proteins and, might be extensively applied in experimental diagnosis, molecular imaging and targeted therapeutics in the future.